Detectable Reporter Gene Expression following Transduction of Adenovirus and Adeno-Associated Virus Serotype 2 Vectors within Full-Thickness Osteoarthritic and Unaffected Canine Cartilage In Vitro and Unaffected Guinea Pig Cartilage In Vivo

Department of Veterinary Clinical Sciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210, USA.
Journal of Orthopaedic Research (Impact Factor: 2.99). 11/2009; 28(2):149-55. DOI: 10.1002/jor.20975
Source: PubMed

ABSTRACT This study quantified and compared the transduction efficiencies of adenoviral (Ad), Arg-Gly-Asp (RGD)-modified Ad, adeno-associated viral serotype 2 (AAV2), and self-complementary AAV2 (scAAV2) vectors within full-thickness osteoarthritic (OA) and unaffected canine cartilage explants in vitro. Intraarticular administration of Ad and scAAV2 vectors was performed to determine the ability of these vectors to transduce unaffected guinea pig cartilage in vivo. Following explant exposure to vector treatment or control, the onset and surface distribution of reporter gene expression was monitored daily with fluorescent microscopy. At termination, explants were divided: one half was digested for analysis using flow cytometry; the remaining portion was used for histology and immunohistochemistry (IHC). Intact articular joints were collected for real-time RT-PCR and IHC to detect reporter gene expression following injection of selected vectors. Ad vector transduced focal areas along the perimeters of explants; the remaining vectors transduced chondrocytes across 100% of the surface. Greater mean transduction efficiencies were found with both AAV2 vectors as compared to the Ad vector (p < or = 0.026). Ad and Ad-RGD vectors transduced only superficial chondrocytes of OA and unaffected cartilage. Uniform reporter gene expression from AAV2 and scAAV2 was detected in the tangential and transitional zones of OA cartilage, but not deeper zones. AAV2 and scAAV2 vectors achieved partial and full-thickness transduction of unaffected cartilage. In vivo work revealed that scAAV2 vector, but not Ad vector, transduced deeper zones of cartilage and menisci. This study demonstrates that AAV2 and scAAV2 are reliable vectors for use in cartilage in vitro and in vivo.

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    • "However, further improvement of defect healing has been achieved by genetically modifying autologous chondrocytes during in vitro culture. Even though adenovirus has gained increasing attention as a tool for efficient delivery of genetic material into chondrocytes (Dinser et al., 2001; Hidaka et al., 2001; Li et al., 2004; Oberholzer et al., 2007; Santangelo et al., 2010), very little is known about the effect of transduction itself. Using a Figure 2. The transduction of human primary chondrocytes with recombinant adenovirus leads to an MOI-dependent increase in matrix production. "
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    ABSTRACT: The present study examines the effects of adenoviral (Ad) transduction of human primary chondrocyte on transgene expression and matrix production. Primary chondrocytes were isolated from healthy articular cartilage and from cartilage with mild osteoarthritis (OA), transduced with an Ad vector and either immediately cultured in alginate or expanded in monolayer before alginate culture. Proteoglycan production was measured using dimethylmethylene blue (DMMB) assay and matrix gene expression was quantified by real-time PCR. Viral infection of primary chondrocytes results in a stable long time transgene expression for up to 13 weeks. Ad transduction does not significantly alter gene expression and matrix production if chondrocytes are immediately embedded in alginate. However, if expanded prior to three dimension (3D) culture in alginate, chondrocytes produce not only more proteoglycans compared to non-transduced controls, but also display an increased anabolic and decreased catabolic activity compared to non-transduced controls. We therefore suggest that successful autologous chondrocyte transplantation (ACT) should combine adenoviral transduction of primary chondrocytes with expansion in monolayer followed by 3D culture. Future studies will be needed to investigate whether the subsequent matrix production can be further improved by using Ad vectors bearing genes encoding matrix proteins.
    Biotechnology and Bioengineering 09/2012; 109(9):2402-8. DOI:10.1002/bit.24505 · 4.13 Impact Factor
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    • "Recombinant E-1 deleted human serotype 5 adenovirus preparations, single-stranded human serotype 2 AAV2, and scAAV2 containing GFP under the control of the cytomegalovirus (CMV) promoter were generated and purified by cesium chloride density gradient method [12] [15]. Particle count of Ad vectors was determined by optical density at 260 nm, and particle titers of rAAV2 and scAAV2 vectors were determined by DNase-resistant particle (DRP) values using real-time PCR assay as described previously [16] "
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    ABSTRACT: Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad), serotype 2 adeno-associated virus vectors (rAAV2), or self-complementary (sc) AAV2 vectors carrying green fluorescent protein (GFP). Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb) titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.
    01/2012; 2012:735472. DOI:10.1155/2012/735472
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